Donor Cells for Cloning—Many Are Called But Few Are Chosen

Abstract

The few viable clones obtained at the end of a typical cloning experiment are genetic copies of the donor cell genome of a nonreproductive (somatic) or embryonic cell used for nuclear transfer. Nuclear totipotency is restored by gene reprogramming which probably involves erasure of epigenetic constraints imposed on the donor genome during differentiation. Reprogramming has to take place in the short interval between entry of the donor genome into the egg cytoplasm and normal onset of embryonic genome activation. It involves active chromatin remodeling and leads to a highly selective switching-off and-on of genes in the nuclear donor at the right time and quantitative level of expression. Less than 5% of the more than 200 histologically distinct mammalian cell types have been tested as nuclear donors and proven to be capable of generating cloned offspring. Therefore, an ideal nuclear donor may have not yet been found. This review focuses on the impact of genetic and epigenetic differences between donor cell types on successful mammalian cloning. Genetic differences include genomic nonequivalence between donor cells, gene damage, mitochondrial heteroplasmy, genetic background and degree of heterozygosity, presence of an inactive X-chromosome, and differences in telomere length. Epigenetic differences arise from changes in gene activity and not in DNA sequence. They lead to varying degrees of cellular differentiation and entail stable changes in chromatin structure, such as covalent modifications of DNA and DNA-binding proteins, preceding transcriptional activation. Differences between donor cell cycle stages can also be referred to as epigenetic and influence the usefulness of a given donor cell genome in nuclear cloning. Finally, this review summarizes practical aspects of nuclear cloning, including current protocols of donor cell isolation, purification, in vitro culture, cell cycle synchronization, and preparation for nuclear transfer.